This invention relates to the field of electrophoresis. More specifically, this invention relates to electrophoretic methods and apparatus useful for the purification of nucleic acids.
Analysis of nucleic acid structure has become the focus of much of modern biology, biotechnology and medicine. Modem nucleic acid analysis techniques such as PCR, fragment-length-polymorphism analysis, and DNA sequencing provide information useful for a variety of applications including diagnosis of disease, organism identification, and tracking evolutionary relatedness. A necessary preliminary step in any nucleic acid analysis method is the preparation of nucleic acid which is free from contaminants which can interfere with enzymes used in these techniques, e.g., contaminants which can inactivate polymerase enzymes used in PCR and DNA sequencing methods.
A wide variety of nucleic acid purification techniques are available based on a range of different physical and chemical principles. The most common nucleic acid purification methods include organic/aqueous liquid-liquid extraction, solid-phase adsorption, precipitation, density-gradient centrifugation, and preparative electrophoresis. Electrophoretic methods are particularly attractive because they result in nucleic acid having a high purity and a large molecular weight.
But, conventional preparative electrophoretic methods suffer from significant shortcomings which limit their practical utility, particularly in the context of high-throughput applications. A particularly problematic aspect of conventional preparative electrophoretic methods is the manner in which a purified nucleic acid is removed from an electrophoresis medium, e.g., an electrophoresis gel. In one class of sample removal processes, the purified nucleic acid is manually excised from the electrophoresis gel. These sample-excision methods are disadvantageous because the nucleic acid and gel material must be separated after excision of the sample band, the procedure requires significant manual intervention, and the purified nucleic acid must be visualized prior to excision in order to locate the desired sample band. In a second class of sample removal techniques, the purified sample is eluted off of the electrophoresis gel into a gel-free buffer. However, such elution methods require that multiple fractions be collected, the purified sample band be visualized, and/or the elution properties of the desired nucleic acid be known.
The present invention is directed towards the discovery of a class of novel preparative electrophoresis methods useful for the purification of nucleic acids. The methods are particularly useful for the preparation of nucleic acid samples prior to treatment with enzymes, e.g., in Sanger-type sequencing, oligonucleotide ligation assays, and PCR.
In a first aspect, the invention comprises a method for purifying a nucleic acid sample comprising the steps of (1) providing a nucleic acid sample comprising a desired nucleic acid and one or more contaminants, (2) providing an electrophoresis matrix having a loading well and a recovery well formed therein, placing the nucleic acid sample into the loading well, (3) performing a first electrophoresis comprising electrophoresing the nucleic acid sample for a first time effective to transport the desired nucleic acid out of the loading well and into the electrophoresis matrix, (4) performing a second electrophoresis comprising electrophoresing the nucleic acid sample for a second time effective to transport the desired nucleic acid out of the electrophoresis matrix and into the recovery well, (5) wherein the first and second electrophoresis is effective to substantially reduce the concentration of contaminants relative the concentration of desired nucleic acid, thereby producing a purified nucleic acid. In the method, the loading well and the recovery well may be the same well or different wells.
In a first preferred embodiment of this first aspect of the invention, referred to herein as the xe2x80x9ctrap modexe2x80x9d of the invention, the loading well and the recovery well are spatially overlapping wells, and the electrophoresis matrix comprises a bulk portion and a well-matrix-interface portion, and the matrix is effective to trap the desired nucleic acid in the well-matrix-interface portion such that the desired nucleic acid is substantially prevented from entering the bulk portion of the matrix.
In a second preferred embodiment of the first aspect, referred to herein as the xe2x80x9ccontaminant-dilution modexe2x80x9d of the invention, the loading well and the recovery well are spatially overlapping wells, and the first electrophoresis is sufficient to transport a portion of the contaminants out of the loading well, through the electrophoresis matrix, and into a contaminant dilution reservoir, the reservoir containing a volume of buffer sufficient to substantially dilute the contaminants entering the reservoir.
In a third embodiment of the first aspect of the invention, referred to herein as the xe2x80x9cLITAC-reverse-field modexe2x80x9d, the loading well and the recovery well are spatially overlapping wells, the first electrophoresis employs a DC electrical field and the second electrophoresis employs a LITAC electrical field.
In a fourth preferred embodiment of the first aspect present invention, the loading and recovery wells are spatially distinct, and the first electrophoresis employs a DC electrical field and the second electrophoresis employs a LITAC electrical field.
In a fifth preferred embodiment of the first aspect present invention, the loading and recovery wells are spatially distinct, and the first electrophoresis comprises electrophoresing the desired nucleic acid in a first direction and the second electrophoresis comprises electrophoresing the desired nucleic acid in a second direction different from the first direction.
In a second aspect, the present invention consists of a method for the electrophoresis of a nucleic acid sample located in an electrophoresis matrix comprising subjecting the nucleic acid sample to a LITAC electrical field comprising a forward electrical field EF and a reverse electrical field ER.
In a third aspect, the present invention comprises a method for the electrophoresis of a nucleic acid sample located in an electrophoresis matrix comprising subjecting the nucleic acid sample to a ZIVE electrical field comprising a forward electrical field EF and a reverse electrical field ER.
Various aspects and/or embodiments of the above-described invention achieve one or more of the following important advantages over known electrophoretic purification methods: (1) using the methods of the invention, there is no need to physically remove sample bands from an electrophoresis gel subsequent to electrophoretic separationxe2x80x94instead, the purified sample is located in a gel-free recovery well and is dissolved in a buffer suitable for subsequent enzymatic treatment, thereby greatly facilitating the automation of post-purification sample recovery; (2) using the methods of the invention, there is no need to collect multiple fractions resulting from a post-electrophoresis elution processxe2x80x94instead, the purified sample is located in a gel-free recovery well and is dissolved in a buffer suitable for subsequent enzymatic treatment, thereby reducing the amount of sample dilution, eliminating the need to collect multiple fractions, and eliminating the requirement for a priori knowledge of the migration behavior of a desired nucleic acid; (3) using the methods of the invention, there is no need to visualize the desired nucleic acid subsequent to electrophoretic separation in order to effect its recoveryxe2x80x94instead, subsequent to electrophoresis, the purified sample is located in a gel-free recovery well; (4) using the methods of the invention, there is no need to separate an electrophoresis matrix from a purified nucleic acid; and, (5) using the methods of the invention, nucleic acid is purified to a sufficient purity to allow for efficient PCR amplification in a single step without the need to perform centrifugation or ethanol precipitation to concentrate the purified nucleic acid.
These and other features and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.